Research On Autotoxins'temporal And Spatial Distribution And Microbial Communities In The Root Zones Of Rehmannia Glutinosa L.

Rehmannia glutinosa Libosch.—belongs to the Scrophulariaceae family,possess precisely clinic effect.It is also one of the most common medicinal herbal plants in the process that industry chain of traditional Chinese medicine were constructed.However,the consecutive monoculture problems in R.glutinosa resulted in tremendous decrease of its yield and quality.To clarify the formation mechanism of this issue,the relationship between phenolic acids were revealed,and the autotoxins spatial distribution as same as microbial communities within R.glutinosa root zones was addressed in this work.The main results were as follows:1.From previous enlargement stage,allelopathic autotoxicity effect was significantly increased and the farthest range of this effect could reach to 20 cm from the plantBioassay test from different growth stages of root exudates of R.glutinosa showed that the radicle length was facilitated from seeding to previous enlargement stage,and a gradually increasing inhibited effect was shown from previous enlargement stage and peaked at harvest time(IR=41%).Moreover,the bioassay test with different root zones soil showed that the inhibited effect was decreased with the increase of distance from R.glutinosa and vanished when the distance was more than 20 cm,suggesting that R.glutinosa was significantly affected by the allelopathic autotoxicity from previous enlargement stage and the diameter of this allelopathic autotoxicity effect was 40cm,the soils of which were under the above-ground of R.glutinosa.2.Syringic acid was the dominated autotoxin of R.glutinosaHPLC results from root exudates of different growth stages showed that 5 phenolic acids(coumalic acid,4-hydroxybenzoic acid,venillic acid,syringic acid,ferulic acid)contents have irregular change with the growth of the plant.The correlation analysis between phenolic acids and radicle length demonstrated that only syringic acid have negatively correlation with allelopathic autotoxicity(r=-0.841,P<0.05).Furthermore,HPLC results of from soils of different root zones showed that 5 phenolic acids,including 4-hydroxybenzoic acid,vanilic acid,syringic acid,3,4-dihydroxy benzoic acid and vanillin)cloud be detected in all samples.The contents of these phenolic acids of the soil inside allelopathic autotoxic circle were significant higher than that of outside that have the same level of with the soil in which R.glutinosa were never been planted,implying syringic acid was the dominated autotoxin and the other phenolic acids might work by synergistic effect.3.Besides phenolic acids,other potential autotoxins of R.glutinosa play important roles in allelopathic autotoxity.Compounds in soils of different root zones were extracted by methanol and n-pentane before GC-MS analysis.The results showed that 20 new compounds were generated inside the allelopathic autotoxicity circle after R.glutinosa were cultivated.Of which,five of them with strong allelopathic effect,including 3-oxo-2-(2-pentenyl)-,methyl ester,[1R-[1a,2b(Z)]]-,1,3-Benzodioxole-5-carb oxaldehyde,4,7-dimethoxy-,(7Z,10Z,13Z)-7,10,13-Hexadecatrienal,(9E)-9-hexadecenoic acid and palmitoleic acid,have been reported.Remain 15 compounds might be new important candidate of autotoxins that is harmful to R.glutinosa g rowth.4.The microbial community in soils of different root zone remarkably changed and some new pathogenic microbes were induced after R.glutinosa cultivating might related to the occurrence of diseases during following cultivation.Microbial community in soils of different root zone was analyzed by PCR-DGGE technology and the profiles of soils microbes showed more bands of inside the allelopathic autotoxity circle than outside.Differential bands were selected and sequenced according to 16S rDNA and 18S rDNA information of microbes.Results showed that these microbes divided into nine phyla,including Bacteroidetes,Chloroflexi,Firmicutes,Gemmatimonadetes,Proteobacteria,Actinobacteria,Ascomycota,Basidiomycota and Fungi incertae sedis.Notable 6 genera that were sihnificant pathogenic to plants,including Acremonium,Humicola,Chitinophaga,Micrococcus,Pseudomonas and Bacillus,were found after R.glutinosa cultivating.A conclusion were predicted these pathogenic microbes were closely related to the occurrence of R.glutinosa diseases under consecutive cultivating mode.To sum up,the stage when allelopathic autotoxicity effect started and the range where this effect can reached were demarcated.Syringic acid was regard as the dominated autotoxin of R.glutinosa.Moreover,we consider that some phenolic acids(coumalic acid,4-hydroxybenzoic acid,venillic acid,ferulic acid,3,4-dihydroxybenzoic acid and vanillin)and other 5 kinds of compounds,incl uding 3-oxo-2-(2-pentenyl)-,methyl ester,[1R-[1a,2b(Z)]]-,1,3-Benzodioxole-5-c arboxaldehyde,4,7-dimethoxy-,(7Z,10Z,13Z)-7,10,13-Hexadecatrienal,(9E)-9-hexa decenoic acid and palmitoleic acid,as potential autotoxins of R.glutinosa.Additionly,we also found 6 kinds of pathogenic microbes that are closely related to the occurrence of diseases during following cultivation.In conclusion,these compounds and pathogenic microbes give insight into understanding formation mechanism of the consecutive monoculture problems of R.glutinosa.